photosynthesis when carbohydrates build up in the mesophyll .... measured the light-limited quantum yield (ΦCO#,app. ) ..... Correia MJ, Chaves MMC, Pereira JS.
RESEARCH New Phytol. (2000), 145, 39–49
Diurnal regulation of photosynthesis in understory saplings E R I C L. S I N G S A A S"*, D O N A L D R . O R T",# E V A N H. D L U C I A" " Department of Plant Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801 USA # Photosynthesis Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Urbana, IL 61801 USA Received 24 May 1999 ; accepted 6 September 1999 Photosynthetic rates of plants grown in natural systems exhibit diurnal patterns often characterized by an afternoon decline, even when measured under constant light and temperature conditions. Since we thought changes in the carbohydrate status could cause this pattern through feedback from starch and sucrose synthesis, we studied the natural fluctuations in photosynthesis rates of plants grown at 36 and 56 Pa CO at a FACE (free# air-CO -enrichment) research site. Light-saturated photosynthesis varied by 40 % during the day and was # independent of the light-limited quantum yield of photosynthesis, which varied little through the day. Photosynthesis did not correspond with xylem water potential or leaf carbohydrate build-up, but rather with diurnal changes in air vapor-pressure deficit and light. The afternoon decline in photosynthesis also corresponded with decreased stomatal conductance and decreased Rubisco carboxylation efficiency which in turn allowed leafairspace CO partial pressure to remain constant. Growth at elevated CO did not affect the afternoon decline in # # photosynthesis, but did stimulate early-morning photosynthesis rates relative to the rest of the day. Plants grown at 56 Pa CO had higher light-limited quantum yields than those at 36 Pa CO but, there was no growth–CO effect # # # on quantum yield when measured at 2 kPa O . Therefore, understory plants have a high light-limited quantum # yield that does not vary through the day. Thus, the major diurnal changes in photosynthesis occur under lightsaturated conditions which may help understory saplings maximize their sunfleck-use-efficiency. Key words : photosynthesis, diurnal variation, elevated CO , FACE, understory, Acer rubrum, Liquidambar # styraciflua, Cercis canadensis.
Under natural conditions, photosynthesis is biochemically regulated to maintain a balance between the rates of its component processes and the concentrations of metabolites (Geiger & Servaites, 1994) while environmental variables such as sunlight, water availability, and atmospheric conditions continuously change. Regulatory mechanisms can respond either to environmental stimuli (exogenous factors) or to biochemical limitations of the mesophyll cells (endogenous factors). Photosynthesis responds to exogenous controls such as excess photon flux density (PFD) (Correia et al., 1990 ; O$ gren & Sjostrom, 1990 ; Watling et al., 1997), drought (Valentini et al., 1995 ; Basu et al., 1998), and leaf-air vapor pressure difference (VPD ; Tenhunen et al., 1984 ; Raschke & Resemann, 1986 ; Pathre et al., 1998), and to endogenous controls such *Author for correspondence (tel j1 217 244 3167 ; fax j1 217 244 7246 ; e-mail singsaas!uiuc.edu).
as feedback limitation by end-product synthesis (Goldschmidt & Huber, 1992 ; Pammenter et al., 1993 ; Bu$ ssis et al., 1997 ; Greer, 1998). These controls are not independent. For example, photosynthesis responds to changes in leaf-air VPD by depressing the biochemistry of photosynthesis in the mesophyll cells (Sharkey, 1984 ; Wise et al., 1991), and to excess PFD, by decreasing the efficiency of electron transport (O$ gren & Sjostrom, 1990 ; Watling et al., 1997), an endogenous process. In this way, interactions between exogenous and endogenous factors determine the diurnal pattern of photosynthesis. This regulation is important for plants growing in a forest understory, where periods of non-saturating PFD are punctuated by sunflecks that can provide nearly full sunlight. This condition presents a problem and an opportunity for photosynthesis in the understory. If sunfleck PFD is greater than the photosystems can use, nonphotochemical quenching reduces the quantum yield of photosynthesis
40
RESEARCH E. L. Singsaas et al.
(Watling et al., 1997). On the other hand, plants that can rapidly induce photosynthesis during a sunfleck can make optimal use of the high PFD. The efficiency with which sunflecks are used is important in determining the daily carbon gain of plants in understory environments (Sims & Pearcy, 1993 ; Pearcy & Yang, 1998). Therefore, the diurnal regulation of photosynthesis can potentially benefit plants most by increasing their sunfleck-useefficiency. Photosynthesis is also sensitive to changes in atmospheric CO partial pressure. Elevated CO # # effects on photosynthesis are well documented, and often include increased maximum photosynthesis rate, reduced leaf Rubisco content, reduced chlorophyll content, increased leaf carbohydrate content, increased quantum yield, and increased water-use and light-use efficiencies (Van Oosten et al., 1994 ; Drake & Gonzalez-Meler, 1996 ; Nakano et al., 1997 ; Osborne et al., 1997 ; Curtis & Wang, 1998 ; Theobald et al., 1998 ; Wu$ rth et al., 1998). Many of these effects are related ; for example the reduced expression of photosynthesis-related genes is signalled by a build-up of carbohydrates resulting from increased photosynthesis rates at elevated CO # (Goldschmidt & Huber, 1992 ; Van Oosten et al., 1994 ; Van Oosten & Besford, 1996 ; Bu$ ssis et al., 1997 ; Wu$ rth et al., 1998). It has been suggested that a similar mechanism can influence the diurnal pattern of photosynthesis through sink limitations of photosynthesis when carbohydrates build up in the mesophyll cells (Sinha et al., 1997 ; Greer, 1998). Our objective was to determine what endogenous and exogenous factors control photosynthesis in understory hardwood trees. We used saplings grown outdoors in the forest-atmosphere-carbon-transferand-storage 1 (FACTS-1) research site, where an intact pine forest is fumigated with CO using free# air-CO -enrichment (FACE) technology to elevate # the atmospheric CO concentration by 20 Pa. We # expected plants grown at elevated CO to accumulate # more carbohydrates throughout the day than those at ambient CO (Rey & Jarvis, 1998 ; Wu$ rth et al., # 1998). Thus the CO treatment served as a ma# nipulation of the carbohydrate status of the leaves, which allowed us to investigate endogenous control via photosynthetic end-product build-up. To examine the endogenous and exogenous controls of photosynthesis, we measured leaf gas-exchange, chlorophyll fluorescence and leaf water potential, and leaf carbohydrate levels repeatedly throughout the day. Study site Measurements were made on understory saplings at the FACTS-I research site in Chapel Hill, NC,
USA (35E 97h N, 79E 09h W). At this site, three 30m-diameter plots were continuously fumigated to raise the atmospheric CO concentration to 20 Pa # above current ambient levels (Hendrey & Kimball, 1994 ; Lewin et al., 1994 ; Hendrey et al., 1999). Three additional fully-instrumented rings were fumigated with compressed air as controls. Fumigation was initiated in August 1996, and all measurements were made in June and August 1998. The plots were established in a loblolly pine plantation which has been unmanaged since its planting in 1983. We selected three deciduous species that had established naturally in the understory : sweetgum (Liquidambar styraciflua L., 811 stems ha−"), eastern redbud (Cercis canadensis L., 160 stems ha−"), and red maple (Acer rubrum L., 170 stems ha−"). Subject trees were 3 m tall. Gas-exchange Leaves of five individuals of each species were tagged at the beginning of each day, and the photosynthetic light-response of each leaf was measured five times during the day (every 2 h beginning at 08.00 hours). Each light-response-curve took 15 min to complete, and there was no evidence of previous measurements affecting the light-response of a leaf (E. H. DeLucia, unpublished). Measurements were made on six consecutive days in August 1998 which had similar weather conditions and were uninterrupted by rain. Photosynthetic CO response (A–Ci # curves) were measured on sweetgum trees three times daily (08.00, 13.00 and 16.00 hours). Photosynthetic measurements were made using an open gas-exchange system (model LI-6400 ; LICOR Inc., Lincoln, NE, USA). Leaves were clamped in the standard 6 cm# cuvette. Light was provided by a red-blue light emitting diode (LED) array, and ambient CO partial pressure (Ca) was # maintained during measurement at 37 Pa for leaves measured in control plots and 57 Pa for leaves measured in treatment plots. Data were logged at 14 light levels between 800 and 0 µmol m−# s−" PFD, and the leaf was given between 45 and 300 s to stabilize. Leaves usually stabilized within 60 s. Leaf temperature was maintained at 25mC and the dewpoint in the gas-exchange cuvette was maintained between 16 and 20mC which maintained a constant leaf-air VPD during measurements. Low-oxygen measurements were made by providing a mixture of 98 % N j2 % O to the inlet port of the system, and # # controlling Ca to 60 Pa. The maximum quantum yield of CO fixation was # calculated by regression of the linear region of the light-response curves (3–6 points ; usually at PFD 60 µmol m−# s−") on an incident-light basis. The maximum rate of RuBP regeneration (Jmax) and of Rubisco carboxylation (Vcmax) were calculated by fitting A–Ci curve data (eight points per curve) to the
RESEARCH Diurnal regulation of photosynthesis equations of Farquhar et al. (1980) by non-linear least-squares regression as in Harley & Tenhunen (1991). Model parameters were from Harley & Baldocchi (1995). Chlorophyll fluorescence Chlorophyll fluorescence was measured following each gas-exchange measurement using a portable pulse-modulated fluorometer (model PAM-2000, Heinz Walz GmbH, Effeltrich, Germany). Maximum photochemical efficiency of PSII (Fv\Fm) was measured in leaves that had been dark adapted for 30 min. Xylem pressure potential Pressure potential (Ψ) was measured with a pressure bomb (model 3005, Soilmoisture Equipment Corp., Santa Barbara, CA, USA) from five trees of each species in each ring. Measurements coincided with gas-exchange and fluorescence measurements.
41
1990). Treatment comparisons of light-saturated photosynthesis rates and quantum yields were made using nested ANOVA where plots are nested within a treatment (elevated CO or control). Linear trends # were compared using ANCOVA as described in Sokal & Rohlf (1995). Statistics were calculated using SYSTAT (SPSS Inc., Chicago, IL, USA). Probability values 0.05 were considered significant. Individual saplings in the same plot were considered statistically independent because the spatial variation in CO within each plot was greater than # the variation between plots (K. F. Lewin, unpublished). Since C. canadensis and A. rubrum saplings did not occur in all plots, five individuals were sampled in each of two plots (one at 36 Pa and one at 56 Pa).
RESULTS Micrometeorology
Leaf carbohydrate analysis Ten 1.05-cm# punches (14–20 mg d. wt) were collected from leaves adjacent to those used for gasexchange and chlorophyll fluorescence measurements. Samples were dried at 70mC for 3 d, and analyzed for non-structural carbohydrates as described in Tissue & Wright (1995). Micrometeorology The temporal variability of meteorological parameters was measured at a single site near the center of a treatment and a control plot. PFD, CO , and # water vapor concentration were sampled at 1-min intervals. Total PFD is defined as the sum of diffuse and direct PFD and was measured with a galliumarsenide photodiode (Hamamatsu Corp., Middlesex, NJ, USA), which was cosine-corrected with a small teflon diffusion-disc and calibrated under sunlight against a quantum sensor (model LI-185, LI-COR, Inc.). Diffuse PFD was measured with a photodiode as already described with a shadow band mounted 6 cm from the surface. To measure CO (data not # reported) and water vapor concentrations, ambient air was drawn by a small pump through a 1.5 m piece of tubing to an IRGA (model 6262, LI-COR, Inc.). All sensors and gas inlets were mounted 1 m above the forest floor. The 5 V outputs from the IRGA and the bridged photodiode output were recorded on a data logger (model CR21x, Campbell Scientific Inc., Logan, UT, USA). Ambient air temperature was logged by the FACTS-I site control system. Statistical analysis Diurnal data were analyzed using repeated-measures ANOVA (CO i time of day ; Potvin & Lechowicz, #
The environmental conditions in both plots were similar, so data from a representative day in an elevated CO plot are shown (Fig. 1). PFD averaged # 121 µmol m−# s−" with a median of 68 µmol m−# s−" between 08.00 and 17.00 hours, and ranged from 18 to 1470 µmol m−# s−". One quarter of the readings were 60 µmol m−# s−" but these provided 75 % of the total daily photon flux. Diffuse PFD during the middle of the day was more normally distributed with a mean of 28.7 and a median of 29.6, ranging from 4.8 to 61.1 µmol m−# s−". Vapor pressure deficit (VPD) exhibited a strong diurnal variation, climbing from near zero in the morning to 1.7 kPa during the middle of the day. The peak in VPD occurred after 16.00 hours which coincided with the maximum daily air temperature.
CO effects on light-limited and light-saturated # photosynthesis To assess whether growth under CO enrichment # affected the photosynthetic response to PFD, we measured the light-limited quantum yield (ΦCO ,app) # and the light-saturated rate of CO assimilation on L. # styraciflua trees between 10.00 and 15.00 hours (Table 1). When measured at the same Ca, ΦCO ,app # did not differ between trees grown at elevated and ambient CO (P l 0.654, n l 18). However, at their # respective growth CO concentration, plants at 56 Pa # CO had 20 % greater quantum yields (0.066 vs # 0.055 ; P 0.001). Light-saturated rates of photosynthesis were 30 % higher for plants grown at elevated CO when measured at the same CO # # conditions as control plants (P l 0.043, n l 18). Comparisons of plants at their respective growth CO shows a 100 % increase in light-saturated #
Total PFD (µmol m–2 s–1)
1500 60 1000
40
500
20
0
0 Air temperature (°C)
Diffuse PFD (µmol m–2 s–1)
RESEARCH E. L. Singsaas et al.
2.0
30
1.5 25
1.0 0.5
20 00.00
VPD (kPa)
42
08.00
16.00
24.00
08.00
0.0 24.00
16.00
Time of day (hours)
Fig. 1. Diurnal variations in diffuse and total (diffuse j direct) irradiance (PFD), air temperature, and vapor pressure deficit (VPD) in the understory at the FACTS-I research site. Data points were collected once per minute on 11 June 1998 and were not averaged or smoothed.
Table 1. Apparent quantum yield (ΦCO ,app) and light-saturated # photosynthesis rates (Asat) of understory Liquidambar styraciflua trees grown at ambient (36 Pa) and elevated (56 Pa) CO # ΦCO
Asat
#,app
Measurement conditions
Grown at ambient CO #
Grown at elevated CO #
Grown at ambient CO #
Grown at elevated CO #
36 Pa CO # 56 Pa CO #
0n055p0n001 0n066p0n002
0n057p0n002 0n066p0n002
3n9p0n43 6n0p0n62
5n1p0n42 7n8p0n61
Measurements were made at 21 KPa O and either 36 or 56 Pa CO . MeanspSE # # are presented.
Table 2. Quantum yield of understory Liquidambar styraciflua, Cercis canadensis and Acer rubrum saplings at the FACTS-I research site 21 kPa O #
2 kPa O #
Species
36 Pa CO #
56 Pa CO #
36 Pa CO #
56 Pa CO #
Liquidambar styraciflua Cercis canadensis Acer rubrum
0n060p0n002 0n063p0n001 0n055p0n002
0n067p0n002 0n072p0n002 0n066p0n002
0n090p0n003 0n093p0n002 0n088p0n003
0n092p0n003 0n098p0n003 0n090p0n005
Measurements were made at ambient CO and O partial pressures (ambient) or at 2 kPa O and 60 Pa CO (low O ). # # # # # All measurements were made in June 1998. Data are reported as meanspSE.
photosynthesis (7.8 vs 3.9 µmol CO m−# s−" ; P # 0.001) for elevated CO grown plants. # To assess whether acclimation to elevated CO # had occurred in all three species under investigation, we measured the light-limited quantum yield on all species at their growth CO with 21 kPa O and at 60 # # Pa CO with 2 kPa O (Table 2). When measured at # # their ambient CO and O , quantum yields were # #
between 10 and 20 % higher at elevated CO for all # three species (P 0.001, n l 36). When we suppressed photorespiration by making measurements at 2 kPa O , we detected no difference in light# limited quantum yield between plants grown at ambient and elevated CO (P l 0.136, n l 36). # Therefore, we found no evidence for acclimation of light-limited quantum yield to elevated CO . #
RESEARCH Diurnal regulation of photosynthesis
43 60
Diurnal variance in photosynthesis
A sat 40
Coefficient of variation (%)
We measured the photosynthetic PFD-response repeatedly to investigate the diurnal patterns in photosynthesis (Fig. 2). There was no change in light response in the light-limited region (i.e. 100 µmol m−# s−"). All five lines diverged in the light-saturated region of photosynthesis. The light-saturated rate of photosynthesis was low at 08.00 hours, and increased until 12.00 hours, at which point it decreased throughout the afternoon. This pattern is similar to that for stomatal conductance. Photosynthesis did not, however, appear to be limited by Ci since the low rate of photosynthesis at 08.00 hours occurred at a Ci similar to the midday values. The decline in Ci in these data is atypical as there was no significant time of day effect on Ci (see below). The coefficient of variation in light-saturated photosynthesis was between 27 and 44 %. We detected no differences
20
0 φ CO2, app 40
20
0
L. s
A (µmol m–2 s–1)
4
12.00 10.00 14.00
2
08.00 16.00
0
Ci (Pa)
40 30 < 13.00
20
14.00 10
tyra
C. c
ciflu
a
ana
A. r
den
sis
ubr
um
Fig. 3. Coefficient of variation in light-saturated- and light-limited- photosynthesis. Gas-exchange measurements were made on three species grown in two CO # treatments (open bars, 36 Pa ; shaded bars, 56 Pa CO ). # The light-saturated rate of photosynthesis (Asat) was measured at 800 mol m-# s-" PFD, and quantum yield (ΦCO ) was calculated as described in the Materials and # Methods section. All measurements were made from 10–20 June, 1998. Bars represent meanspSE (n l 5).
between the CO treatments (Fig. 3 ; P l 0.62, n l # 24). The coefficient of variation in quantum yield averaged 7 %, and there was no detectable CO effect # (P l 0.11, n l 24).
16.00
gs (mol m–2 s–1)
Diurnal patterns in light-saturated photosynthesis 0.04
12.00 10.00 14.00 08.00
0.02
16.00 0.00 0
300 600 PPFD (µmol m–2 s–1)
Fig. 2. Light-response of photosynthesis (A), leaf-airspace CO partial pressure (Ci) and stomatal conductance (gs) at # five times during the day. Values were calculated from gasexchange measurements made on a single Liquidambar styraciflua leaf at five times during the day. Data were collected on 18 June 1998. Symbols represent curves measured at different times of day and numbers to the right specify the time at which each curve was measured.
Photosynthesis varied diurnally in all three species (Fig. 4 ; P 0.001 for all three species). In most cases, the highest rates occurred at noon and declined thereafter. Photosynthesis was higher at 08.00 hours, relative to the rest of the day, for plants in elevated rather than ambient CO . Photosynthesis rates did # not correspond with the diurnal changes in leaf water potential. Water potential was generally highest at 08.00 hours and declined through the day, but the decline was large only in C. canadensis. There was no CO effect on leaf water potentials during most of the # day, except that in L. styraciflua and C. canadensis leaf water potentials were slightly lower in plants grown at ambient CO during the afternoon (time of # day by treatment interaction ; P l 0.007). Diurnal variation in stomatal conductance (Fig. 5) corresponded to some extent with photosynthesis
44
RESEARCH E. L. Singsaas et al.
A (% of maximum)
L. styraciflua
C. canadensis
A. rubrum
0.9
0.6
0.3
0.0
Ψl (MPa)
– 0.5 – 1.0 – 1.5 – 2.0 08.00 10.00 12.00 14.00 16.00 08.00 10.00 12.00 14.00 16.00 08.00 10.00 12.00 14.00 16.00
Time of day (hours)
Fig. 4. Diurnal patterns of photosynthesis (A) and leaf water potential (Ψ) in three species of understory seedlings. Gas-exchange measurements were made repeatedly on each leaf as described in the Materials and Methods section. Water potential measurements was measured from the same trees, but different leaves were used each time. Measurements were made on trees grown at 36 Pa (open circles) and 56 Pa (closed circles) CO . # Data points represent meanspSE (n l 10 for Liquidambar styraciflua, n l 5 for Cercis canadensis and Acer rubrum). L. styraciflua
C. canadensis
A. rubrum
0.20 gs (mol m–2 s–1)
0.15 0.10 0.05 0.00
Ci (Pa)
400
200
– 2.0 08.00 10.00 12.00 14.00 16.00 08.00 10.00 12.00 14.00 16.00 08.00 10.00 12.00 14.00 16.00
Time of day (hours)
Fig. 5. Diurnal patterns of stomatal conductance (gs) and leaf-airspace CO partial pressure (Ci). Gas-exchange # measurements were made repeatedly on each leaf as described in the Materials and Methods section. Measurements were made on trees grown at 36 Pa (open circles) and 56 Pa (closed circles) CO . Data points # represent meanspSE (n l 10 for Liquidambar styraciflua, n l 5 for Cercis canadensis and Acer rubrum).
(Fig. 4). The variation was significant for L. styraciflua and C. canadensis (P l 0.016, n l 18 ; P 0.001, n l 10) but not for A. rubrum (P l 0.584, n l 7). The co-varying of photosynthesis and stomatal conductance resulted in Ci remaining relatively constant throughout the day for all three
species (L. styraciflua : P l 0.06, n l 18 ; C. canadensis : P l 0.498, n l 10 ; A. rubrum : P l 0.380, n l 7). Because of its higher stomatal conductance, Ci was higher in C. canadensis than in the other two species. To investigate the potential involvement of photo-
RESEARCH Diurnal regulation of photosynthesis L. styraciflua
45
C. canadensis
A. rubrum
Fv/Fm
0.85
0.80
0.75
φCO2
0.09
0.06
0.03
0.00 08.00 10.00 12.00 14.00 16.00 08.00 10.00 12.00 14.00 16.00 08.00 10.00 12.00 14.00 16.00
Time of day (hours)
Diurnal patterns in leaf carbohydrate content To determine whether the build-up of photosynthetic end products in the mesophyll can influence the patterns in photosynthesis rates, we measured soluble and insoluble leaf carbohydrates
20
10
0 60 Jmax (µmol e– m–2 s–1)
inhibition in the diurnal pattern of photosynthesis, we measured the maximum photochemical efficiency of PSII, Fv\Fm, and the maximum light-limited quantum yield of CO fixation, ΦCO (Fig. 6). # # Although Fv\Fm declined steadily throughout the day in two of the three species (L. styraciflua : P 0.001, n l 7 ; C. canadensis : P l 0.014, n l 8), the changes were relatively small. There was no detectable decline in A. rubrum (P l 0.098, n l 7). Light limited quantum yield did not vary diurnally in A. rubrum or C. canadensis (C. canadensis : P l 0.404, n l 8 ; A. rubrum : P l 0.215, n l 7). The statistically significant decline in ΦCO in L. # styraciflua (P 0.001, n l 7) was 10 % at elevated CO and 20 % at ambient CO . In all cases, # # declines were linear throughout the day. To investigate the relationship between photosynthesis and Rubisco kinetics throughout the day, we calculated Vcmax, which estimates the total activity of Rubisco, and Jmax, which estimates the maximum electron transport rate, using A–Ci curves collected from L. styraciflua. Vcmax decreased throughout the day (Fig. 7 ; P l 0.01, n l 8), and there was a significant afternoon decline in Jmax (P l 0.002, n l 8).
Vcmax (µmol CO2 m–2 s–1)
Fig. 6. Diurnal patterns in maximum photochemical yield (Fv\Fm) and the maximum light-limited quantum yield of CO fixation (ΦCO ) . Fv\Fm was measured repeatedly on leaves after a 30 min dark acclimation. # # ΦCO , was calculated from the initial slope of photosynthesis vs PFD response curves measured by gas # exchange. Measurements were made on trees grown at 36 Pa (open circles) and 56 Pa (closed circles) CO . Data # points represent meanspSE (n l 4 for all species).
40
20
0
08.00 12.00 16.00 Time of day (hours)
Fig. 7. Diurnal patterns in maximum carboxylation rate (Vcmax) and maximum electron transport rate (Jmax) estimated from CO -response (A–Ci) curves. Gas# exchange measurements were made repeatedly on leaves of Liquidambar styraciflua growing at 36 Pa (open circles) and 56 Pa (closed circles) CO . Parameters were estimated # from gas-exchange measurements as described in the Materials and Methods section. Data points represent meanspSE (n l 5).
46
RESEARCH E. L. Singsaas et al.
12
8
4
L. styraciflua Total carbohydrates (% d. wt)
0 12
8
4
C. canadensis 0 12
8
4
A. rubrum 0 08.00 12.00 16.00 Time of day (hours)
Fig. 8. Diurnal accumulation of nonstructural carbohydrates in leaves of saplings growing at 36 Pa (open circles) and 56 Pa (closed circles) CO . Leaf punches were # collected three times per day and analyzed for carbohydrates. Data points represent meanspSE. Linear regression was used to fit lines.
(Fig. 8). Leaves grown at elevated CO contained # more total carbohydrates than controls in L. styraciflua (P l 0.007, n l 24) and A. rubrum (P l 0.004, n l 24), but not C. canadensis (P l 0.482, n l 24). Although there was significant diurnal accumulation of carbohydrates in A. rubrum leaves (P l 0.024), we detected no difference in the slopes of the lines (P 0.6), indicating that there was no difference in accumulation rate between elevated CO and control plants. # Light-saturated photosynthesis rates in understory saplings varied by as much as 40 % throughout the day. This percentage change is similar to plants grown in full sunlight (Correia et al., 1990 ; Wise et al., 1991 ; Barton & Gleeson, 1996 ; Sinha et al., 1997 ; Greer, 1998 ; Pathre et al., 1998). However, the diurnal pattern of photosynthesis (low in the
morning and afternoon, with maximum rates at noon) was only seen in one other study to our knowledge (Barton & Gleeson, 1996), with most showing a decline between 12.00 and 14.00 hours. In this study, low photosynthetic rates coincided with decreasing Rubisco carboxylation efficiency and electron transport rates (Fig. 7), both of which are associated with the afternoon decline in photosynthesis (Raschke & Resemann, 1986 ; Wise et al., 1991 ; Geiger & Servaites, 1994 ; Greer, 1998, Tenhunen et al., 1984). These results indicate that care must be taken when interpreting photosynthesis measurements made in the field. Light-saturated photosynthesis rates can vary as much as 40–60 % during the day. This is substantially larger than the 30 % variation in light-saturated photosynthesis attributable to the CO treatment (e.g. Table 1). This variation can be # caused by several factors ; including stomatal conductance, mesophyll carbon reduction capacity, and electron transport capacity, all of which can be influenced by environmental factors such as light and VPD. Gas-exchange measurements by themselves do not provide enough information to deduce which, if any, of these mechanisms is controlling photosynthesis. Some or all of these factors may explain the apparent discrepancy between the apparent upregulation of light-saturated photosynthesis at elevated CO (Table 1) and the apparent # decrease in Vcmax and Jmax (Fig. 7). The low Amax in the ambient CO rings is associated with low stomatal # conductance which corresponds with low mesophyll capacity for photosynthesis. Thus the apparent CO # effect on Amax may be attributed to diurnal regulation rather than acclimation to elevated CO . According # to our results, comparisons between treatments will be most accurate when data are collected during the middle hours of the day (between approx. 10.00 and 15.00 hours), when light-saturated photosynthesis rates are relatively constant. However, it is likely that this period is different for each field site, time of year and species, and must be evaluated on a case-by-case basis. While the overall pattern of photosynthesis throughout the day was not significantly affected by the CO treatment, there was a stimulation of early # morning photosynthetic rates at elevated CO in all # three species (Fig. 4). In C. canadensis, this pattern appears to be associated with elevated stomatal conductance and slightly elevated Ci (Fig. 5), but this is not evident in the other two species. Likewise, our fluorescence data and A–Ci curves do not provide any insight into this phenomenon. This phenomenon may be potentially important in understanding what controls daily carbon gain in an elevated CO # environment and further investigations in this area are warranted. While there was significant diurnal variation in light-saturated photosynthesis, there was little vari-
RESEARCH Diurnal regulation of photosynthesis ation in light-limited photosynthesis. The coefficient of variation in light-limited ΦCO was c. 7 % in all # species. The slight diurnal decline in Fv\Fm suggests that increases throughout the day, as has been suggested in willow canopies (O$ gren & Sjostrom, 1990). However, the declines were small compared with those in the willow studies or in studies demonstrating photoinhibition in rainforest understory species (Watling et al., 1997). We conclude that the changes in Fv\Fm and ΦCO ,app # contributed only marginally to the diurnal patterns in photosynthesis. We calculated the light-limited quantum yield of photosynthesis (ΦCO ,app) in the different CO # # treatments. The only difference we found was caused by suppression of photorespiration during the measurement. This is demonstrated by the lack of detectable differences in ΦCO ,app between control # and treatment plants when measured under the same CO (Table 1) or low O (Table 2) conditions. This # # is consistent with expectations that elevated CO # reduces photorespiration during the measurements without long-term acclimation of ΦCO (Osborne et # al., 1997). Our measurements indicate that ΦCO ,app # is quite high for these plants. Assuming the absorptance (α) of the leaves is near 0.85, increasing ΦCO by c. 20 % brings ΦCO near the maximum # # theoretical value of 0.125. Quantum yields this high have been reported in many species in different studies (Demmig & Bjo$ rkman, 1987 ; Long et al., 1993). While growth at elevated CO is expected to # elicit changes in N investment between carbon reduction and electron transport reactions of photosynthesis (Medlyn, 1996), these would not be detected with measurements of ΦCO because it is, # by definition, measured under light-limiting conditions. Since ΦCO ,app is the product of ΦCO and # # α, its constancy indicates no change in either parameter which is consistent with previous reports (Osborne et al., 1997). Greater investment in electron transport components could increase the range of PFD over which photosynthesis increases linearly. This effect, however, would be masked by the diurnal patterns in stomatal conductance and mesophyll photosynthetic capacity (Figs 2, 5 and 7) making determination of electron transport acclimation difficult. To test for endogenous control of photosynthesis by carbohydrates, we used the CO enrichment # experiment at the FACTS-I site to manipulate the carbohydrate content in leaves of plants growing in a forest. Build-up of carbohydrates can induce changes in the photosynthetic capacity of the mesophyll. Most notably, excess carbohydrates can suppress the expression of photosynthesis-related genes, including Rubisco small subunit which causes a reduction in Rubisco protein (Van Oosten et al., 1994 ; Bu$ ssis et al., 1997). Although this is not likely to play a significant role in the diurnal variation in photo-
47
synthesis, it is partially responsible for the decreased Vcmax of plants grown at elevated CO (Bu$ ssis et al., # 1997). Furthermore, excess carbohydrates can influence photosynthesis directly by increasing the probability of feedback on photosynthesis by reducing the rates of sucrose and starch synthesis (Pammenter et al., 1993). Two of the species we studied, L. styraciflua and A. rubrum, had significantly higher leaf carbohydrate contents at elevated than ambient CO . Because the diurnal patterns in # photosynthesis and stomatal conductance are similar between all three species, it is unlikely that leaf carbohydrate content influenced photosynthesis patterns. The expected effect of growth at elevated CO , a decrease in Vcmax, occurred in L. styraciflua # leaves. Thus, photosynthesis was influenced by an endogenous factor, but there was no evidence of this endogenous influence on the diurnal pattern of photosynthesis. The coincident decline in photosynthesis with the highest VPD suggests that this may be an important signal of the afternoon decline of photosynthesis. Raschke & Resemann (1986) found that increases in VPD throughout the day depressed photosynthetic rates in a Mediterranean shrub. Responses to VPDs may be mediated either by mesophyll sensitivity to the rate of transpiration (Sharkey, 1984) or stomatal closure in response to leaf-to air vapor pressure difference (Yong et al., 1997). In both cases, the VPD response included reductions in the carboxylation efficiency of the mesophyll, an effect commonly associated with the midday decline in photosynthesis (Tenhunen et al., 1984 ; Wise et al., 1991 ; Geiger & Servaites, 1994). This explanation is most consistent with our observations. The maximum VPD we observed occurred later in the day and coincided with the afternoon decline in photosynthesis. Most of the time, light in the understory is diffuse and non-saturating for photosynthesis, the constancy of light-limited photosynthesis indicates that plants assimilate CO slowly and continuously throughout # the day. However, sunflecks allowed leaves to be exposed to as much as 1500 µmol photons m−# s−". This could potentially influence the diurnal pattern of photosynthesis. Plants grown in deep understory shade can become photoinhibited if they receive prolonged full PFD, which would be in excess of what they can use (Watling et al., 1997). For example, light stress has been implicated in the afternoon decline in photosynthesis because of an observed drop in light-limited quantum yield during the afternoon (Correia et al., 1990) and a correlation between daily photon interception and photoinhibition (O$ gren & Sjostrom, 1990). Our data indicate that photoinhibition did not play a strong role in determining the diurnal pattern of photosynthesis in our system. The efficiency of photosynthesis, determined by the light-limited quantum
48
RESEARCH E. L. Singsaas et al.
yield of CO assimilation, were high in all three # species and remained relatively constant throughout the day. Likewise, Fv\Fm did not decrease substantially, and light-saturated photosynthesis rates were highest at midday when sunflecks were most common in the understory. On the other hand, sunflecks provided 75 % of the daily PFD, so diurnal changes in sunfleck use efficiency could also influence daily carbon gain (Sims & Pearcy, 1993 ; Pearcy & Yang, 1998). This is controlled primarily by the induction state of carbonreduction enzymes such as Rubisco (Sassenrath Cole & Pearcy, 1994 ; Krall et al., 1995), and stomatal conductance of the leaf just before the sunfleck (Lei & Lechowicz, 1997 ; Valladares et al., 1997). We observed diurnal patterns in both of these traits. Furthermore, both traits were highest during the middle part of the day, when sunflecks occurred most frequently. During the afternoon, photosynthesis and stomatal conductance declined together, which allowed Ci to remain constant. The maintenance of a high Ci is also thought to improve sunfleck-use-efficiency by maintaining photosynthetic carbon reduction enzymes in their induced states (Krall et al., 1995 ; Lei & Lechowicz, 1997). The diurnal patterns in photosynthesis observed in these understory trees maximized their sunfleck-use efficiency, a trait that correlates with seedling survival in the understory. We would like to thank A. Ward, A. Singsaas and J. Hamilton for assistance in the field and laboratory. We also thank C. Bernacchi, K. George, J. Hartz and H. Maherali for their critical review of the manuscript. This work was supported in part by an Integrative Photosynthesis Research Training Grant from the Department of Energy (no. DEFGO2–92ER20095), funded under the Program for Collaborative Research in Plant Biology. The FACTS1 site was supported by grants from TECO-DOE (DEFG05–95ER62083), NASA (TE\97-0024) and a grant from the United States Department of Energy, Office of Health and Environmental Research, Ecological Research Division.
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